Journal: Fibrogenesis & Tissue Repair

Article Title: Impaired Cav-1 expression in SSc mesenchymal cells upregulates VEGF signaling: a link between vascular involvement and fibrosis

doi: 10.1186/1755-1536-7-13

Figure Lengend Snippet: Impaired Cav- 1/ VEGFR2 co- localization in SSc- MSCs. (A) Immunofluorescence staining of Cav-1 (green) and VEGFR2 (red) in MSCs from HC and SSc. In unstimulated HC-MSCs, Cav-1 (2) is aggregated in some areas of the cell surface while the VEGFR2 (1) is ubiquitously diffuse. After treatment with VEGF both VEGFR2 and Cav-1 (4-6) were co-localized. In the SSc-MSCs, we observed an impaired VEGFR2/Cav-1 co-localization (7-12), after VEGF treatment. Pictures are representative of all experiments. Original magnification 20X. (B) Cav-1 was immunoprecipitated (IP) and its association with VEGFR2 was assessed by western blot (WB). The immunoprecipitation assay showed a lack of that Cav-1/VEGFR2 co-localization in SSc-MSCs. Each experimental condition was performed in triplicate. Blot was representative of all the experiments. Co-immunoprecipitated protein bands were quantified by densitometry and the values were expressed as arbitrary unit of optical density (OD) (** P <0.001).

Article Snippet: Specific rabbit anti-Cav-1 and anti-VEGFR2 antibodies (Santa Cruz Biotechnology, CA, USA) was added and rocked at 4°C for 1 h; 30 μL protein A/G beads (Santa Cruz) was added and the sample was rocked over night at 4°C.

Techniques: Immunofluorescence, Staining, Immunoprecipitation, Western Blot

Journal: Fibrogenesis & Tissue Repair

Article Title: Impaired Cav-1 expression in SSc mesenchymal cells upregulates VEGF signaling: a link between vascular involvement and fibrosis

doi: 10.1186/1755-1536-7-13

Figure Lengend Snippet: Impaired ubiquitination of VEGFR2 and increased VEGF signaling in SSc - MSCs. (A) VEGFR2 was immunoprecipitated (IP) and its association with ubiquitin was assessed by western blot (WB). The immunoprecipitation assay showed that in HC-MSCs, after VEGF treatment, the VEGFR2 immunoprecipitated complexes contained ubiquitin. In SSc-MSCs a decreased level of Ubiquitin associated to the VEGFR2 was observed. (B) Proteins immunoprecipitated (IP) with VEGFR2 antibodies were fractionated by SDS-polyacrylamide gel electrophoresis. Immunoblots were probed with an antibody to phosphotyrosine (pY) and p85. After VEGF treatment, the tyrosine phosphorylation of VEGFR2 in SSc-MSCs was markedly increased when compared to HC-MSC. (C) Western blot of phosphorylated Akt and total Akt. In SSc-MSCs, after VEGF treatment, the levels of p-Akt were higher when compared to HC. Each experimental condition was performed in triplicate. The blots in A-B-C were representative of all the experiments. Protein bands were quantified by densitometry and the values were expressed as arbitrary unit of optical density (OD) (*** P <0.0001).

Article Snippet: Specific rabbit anti-Cav-1 and anti-VEGFR2 antibodies (Santa Cruz Biotechnology, CA, USA) was added and rocked at 4°C for 1 h; 30 μL protein A/G beads (Santa Cruz) was added and the sample was rocked over night at 4°C.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Polyacrylamide Gel Electrophoresis, Phospho-proteomics

Journal: Fibrogenesis & Tissue Repair

Article Title: Impaired Cav-1 expression in SSc mesenchymal cells upregulates VEGF signaling: a link between vascular involvement and fibrosis

doi: 10.1186/1755-1536-7-13

Figure Lengend Snippet: Downregulation of Cav - 1 in HC - MSCs by Cav - 1 - siRNA impaired VEGF signaling. (A) HC-MSC was transfected with specific Cav-1-siRNA (siRNA) or non-targeting siRNA (NT), and Cav-1 protein expression was evaluated by western blot. The cells transfected with Cav-1-siRNA showed a decreased protein expression of Cav-1 when compared with cells transfected with NT siRNA. (B) Proteins immunoprecipitated (IP) by VEGFR2 antibodies were fractionated by SDS-polyacrylamide gel electrophoresis. The immunoblots were probed, by using specific antibodies against phosphotyrosine (pY) and p85. In siRNA HC-MSCs, after VEGF treatment, the tyrosine phosphorylation of VEGFR2 was significantly increased when compared to NT MSCs. (C) Western blot of CTGF expression. VEGF stimulation significantly increased CTGF proteins levels in siRNA HC-MSCs, when compared to NT HC-MSCs. Each experimental condition was performed in triplicate. Blot was representative of all the experiments. The values were expressed as arbitrary unit of optical density (OD) (* P <0.01; ** P <0.001; *** P <0.0001).

Article Snippet: Specific rabbit anti-Cav-1 and anti-VEGFR2 antibodies (Santa Cruz Biotechnology, CA, USA) was added and rocked at 4°C for 1 h; 30 μL protein A/G beads (Santa Cruz) was added and the sample was rocked over night at 4°C.

Techniques: Transfection, Expressing, Western Blot, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Phospho-proteomics

Journal: Oncotarget

Article Title: Tubeimoside-1 suppresses tumor angiogenesis by stimulation of proteasomal VEGFR2 and Tie2 degradation in a non-small cell lung cancer xenograft model

doi: 10.18632/oncotarget.6676

Figure Lengend Snippet: a. Western blot analysis of VEGFR2, Tie2, p-AKT/AKT and p-mTOR/mTOR expression of eEND2 cells (% of control at each time point), which were treated for 0.5h (white circles), 1h (light grey circles), 2h (dark grey circles) and 3h (black circles) with vehicle (0μM; control), 5 and 10μM TBMS1. The data were quantified from 3 independent experiments. Means ± SEM. *P<0.05 vs. vehicle. b. Western blot analysis of VEGFR1, Tie1, VEGFR2 and Tie2 expression of eEND2 cells (% of control), which were treated for 3h with vehicle (0μM; control) or 10μM TBMS1. The data were quantified from 3 independent experiments. Means ± SEM. *P<0.05 vs. vehicle.

Article Snippet: Subsequently, 2μm-thick sections were cut and stained with a rat monoclonal anti-CD31 antibody (1:30; Dianova GmbH, Hamburg, Germany), a rabbit polyclonal anti-VEGFR2 antibody (1:100; Cell Signaling Technology, Frankfurt am Main, Germany) or a goat polyclonal anti-Tie2 antibody (1:100; R&D Systems, Wiesbaden, Germany) followed by a goat-anti-rat IgG Alexa Fluor488-labeled (1:50; Life Technologies, Darmstadt, Germany), a Cy3-conjugated goat-anti-rabbit IgG secondary antibody (1:50; Dianova GmbH) or a Cy3-conjugated donkey-anti-goat IgG secondary antibody (1:50; Dianova GmbH).

Techniques: Western Blot, Expressing, Control

Journal: Oncotarget

Article Title: Tubeimoside-1 suppresses tumor angiogenesis by stimulation of proteasomal VEGFR2 and Tie2 degradation in a non-small cell lung cancer xenograft model

doi: 10.18632/oncotarget.6676

Figure Lengend Snippet: a-b. Relative VEGFR2 (a) and Tie2 (b) mRNA expression of eEND2 cells, which were treated with 10μM TBMS1 for 0, 1, 2 and 3h, as assessed by quantitative real-time PCR. The data were quantified from 3 independent experiments. Means ± SEM. *P<0.05 vs. 0h. c. Western blot analysis of VEGFR2 and Tie2 expression of eEND2 cells (% of time point 0h), which were treated with vehicle (white circles) or 10μM TBMS1 (black circles) in the presence of 100μM CHX for 0, 0.5, 1, 2, 4 and 6h. The data were quantified from 3 independent experiments. Means ± SEM. *P<0.05 vs. vehicle.

Article Snippet: Subsequently, 2μm-thick sections were cut and stained with a rat monoclonal anti-CD31 antibody (1:30; Dianova GmbH, Hamburg, Germany), a rabbit polyclonal anti-VEGFR2 antibody (1:100; Cell Signaling Technology, Frankfurt am Main, Germany) or a goat polyclonal anti-Tie2 antibody (1:100; R&D Systems, Wiesbaden, Germany) followed by a goat-anti-rat IgG Alexa Fluor488-labeled (1:50; Life Technologies, Darmstadt, Germany), a Cy3-conjugated goat-anti-rabbit IgG secondary antibody (1:50; Dianova GmbH) or a Cy3-conjugated donkey-anti-goat IgG secondary antibody (1:50; Dianova GmbH).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oncotarget

Article Title: Tubeimoside-1 suppresses tumor angiogenesis by stimulation of proteasomal VEGFR2 and Tie2 degradation in a non-small cell lung cancer xenograft model

doi: 10.18632/oncotarget.6676

Figure Lengend Snippet: Western blot analysis of VEGFR2 (a, c) and Tie2 (b, d) expression of eEND2 cells (% of control). The cells were pretreated without or with 30μM MG132 or 100μM CQ for 2h, and then incubated with vehicle (distilled water) or 10μM TBMS1 in the presence of 100μM CHX for another 1h (a, c) or 4h (b, d). The data were quantified from 3 independent experiments. Means ± SEM. *P<0.05 vs. vehicle + CHX (control); # P<0.05 vs. TBMS1 + CHX.

Article Snippet: Subsequently, 2μm-thick sections were cut and stained with a rat monoclonal anti-CD31 antibody (1:30; Dianova GmbH, Hamburg, Germany), a rabbit polyclonal anti-VEGFR2 antibody (1:100; Cell Signaling Technology, Frankfurt am Main, Germany) or a goat polyclonal anti-Tie2 antibody (1:100; R&D Systems, Wiesbaden, Germany) followed by a goat-anti-rat IgG Alexa Fluor488-labeled (1:50; Life Technologies, Darmstadt, Germany), a Cy3-conjugated goat-anti-rabbit IgG secondary antibody (1:50; Dianova GmbH) or a Cy3-conjugated donkey-anti-goat IgG secondary antibody (1:50; Dianova GmbH).

Techniques: Western Blot, Expressing, Control, Incubation

Journal: Oncotarget

Article Title: Tubeimoside-1 suppresses tumor angiogenesis by stimulation of proteasomal VEGFR2 and Tie2 degradation in a non-small cell lung cancer xenograft model

doi: 10.18632/oncotarget.6676

Figure Lengend Snippet: a, b. Immunohistochemical detection of VEGFR2 (a, red) and Tie2 (b, red) of CD31-positive microvessels (a, b, green) in a NCI-H460 flank tumor from a vehicle-treated control mouse and a TBMS1-treated animal. Sections were stained with Hoechst 33342 to identify cell nuclei (blue). Arrowheads indicate microvessels with reduced VEGFR2 or Tie2 expression. Scale bars: 35μm. c, d. Quantification of the signal area (% CD31 area) (c) and signal intensity (% CD31 intensity) (d) of microvascular VEGFR2 and Tie2 expression in vehicle-treated and TBMS1-treated tumors. The data were quantified from 8 mice per group. Means ± SEM. *P<0.05 vs. vehicle.

Article Snippet: Subsequently, 2μm-thick sections were cut and stained with a rat monoclonal anti-CD31 antibody (1:30; Dianova GmbH, Hamburg, Germany), a rabbit polyclonal anti-VEGFR2 antibody (1:100; Cell Signaling Technology, Frankfurt am Main, Germany) or a goat polyclonal anti-Tie2 antibody (1:100; R&D Systems, Wiesbaden, Germany) followed by a goat-anti-rat IgG Alexa Fluor488-labeled (1:50; Life Technologies, Darmstadt, Germany), a Cy3-conjugated goat-anti-rabbit IgG secondary antibody (1:50; Dianova GmbH) or a Cy3-conjugated donkey-anti-goat IgG secondary antibody (1:50; Dianova GmbH).

Techniques: Immunohistochemical staining, Control, Staining, Expressing